The present invention relates to cyclic peptido-mimetic compounds, in particular to cyclic peptido-mimetic compounds having azabicycloalkane structure and containing the RGD (Arg-Gly-Asp) sequence. Said compounds have inhibiting action on xcex1vxcex23-receptor of the integrin family. The compounds of the present invention are endowed with antiangiogenic properties, hence are useful as medicaments, preferably for the treatment of tumors.
The first molecule with antiangiogenic activity was discovered in 1975 by Henry Brem and Judah Folkman in cartilaginous tissues.
In the 80s it was found that interferon (xcex1/xcex2) is effective in inhibiting tumor angiogenesis.
In 1998, it was widely published, also in the media, that angiostatin and endostatin discovered by J. Folkman at Harvard Medicinal School and Boston Children""s Hospital were giving very encouraging results in tumor treatment.
To-date, about 30 molecules are tested in clinical trials (Phase I-III).
Of these 30 molecules, only two drugs, of which one is an antibody, are in clinical trials for their activity in inhibiting endothelial specific integrins.
It is calculated that only in the USA, about 9 million patients could benefit from an antiangiogenic therapy.
Recently, FDA has approved clinical trials for the combination of IL-10 with Thalidomide and Methoxyestradiol.
Angiogenesis is intended as the formation of new capillary blood vessels. This natural phenomenon is involved both in physiological processes, as reproduction, and in pathological occurrences, as wound healing, arthritis and tumor vascularization.
A number of growth factors have been identified as capable of promoting angiogenesis, through direct induction of proliferation and/or chemiotaxis of endothelial cells. Other factors, instead, act indirectly, by stimulating other cell types (mast cells, macrophages), which, on their turn, produce angiogenic factors. The presence of growth factors, such as bFGF and VEGF, near a resting capillary net, suggested that angiogenesis might be the outcome of an unbalance between pro-and anti-angiogenic factors.
In the last years, it was reported that tumor growth and metastasis formation is strictly dependent on the development of new vessels capable of vascularizing the tumor mass.
Antiangiogenic tumor therapy is strongly desired by physicians for the following reasons:
specificity: tumor neovascularization is the target;
bioavailability: the antiangiogenic agent is targeted toward endothelial cells, easily reached without the well-known problems of chemotherapy, which is directed on the tumor cell;
chemoresistance: this is the most striking advantage, in fact, endothelial cells are genetically stable and it is quite difficult to observe drug resistance;
angiogenic blockade avoids metastatic cells to diffuse through blood circulation;
apoptosis: blocking angiogenesis makes tumor cell suffer from oxygen and nutrition lack, thus inducing apoptosis;
antiangiogenic therapy does not give rise to side effects typical of chemotherapy.
The endogenous pro-angiogenic factors to date known are acid/basic Fibroblast Growth factor (a/bFGF) and Vascular Endothelial Growth Factor (VEGF), and its subtype B and C, Angiogenin, Endothelial Growth Factor (EGF), Platelet derived-Endothelial Cell Growth Factor (PD-ECGF), Transforming Growth Factor-xcex1 (TGF-xcex1), Transforming Growth Factor-xcex2 (TGF-xcex2), Tumor Necrosis Factor-xcex1 (TNF-xcex1).
Retinoids are tested as potential antiangiogenic agents.
Some PK-C inhibitors, such as Calphostin-C, phorbol esters and Staurosporin, can block angiogenesis, either partially or totally.
Integrins are a class of receptors involved in the mechanism of cell adhesion and alterations in the function of these receptors are responsible in the occurrence of a number of pathologic manifestations, for example embryogenic development, blood coagulation, osteoporosis, acute renal failure, retinopathy, cancer, in particular metastasis. Among the molecular targets involved in angiogenesis, xcex1vxcex23 integrins play an important role in adhesion, motility, growth and differentiation of endothelial cells. xcex1vxcex23 integrins bind the RGD sequence (Arg-Gly-Asp), which constitutes the recognition domain of different proteins, such as laminin, fibronectin and vitronectin. The RGD sequence represent the minimal amino acid domain, in several extra-cellular matrix proteins, which has been demonstrated to be the binding site of the transmembrane integrins proteins family (G. Bazzoni, E. Dejana and M. G. Lampugnani. 1999, Current Opinion in Cell Biology; (11) pp. 573-581). Indeed replacement of just one single amino acid of this short sequence result in loss of binding activity to integrins (F. E. Ali, R. Calvo, T. Romoff, I. Samanen, A. Nichols. 1990, Peptides: Chemistry. Structure and Biology (Eds: J. E. Rivier, G. R. Marshall) ESCOM Science Leiden (Netherland) pp. 94-96). In the last years has been demonstrated that RGD peptide isolated from phage peptides library or biochemically synthesised, were able to compete with extracellular matrix proteins to bind integrins (R. Haubner, D. Fisinger and H. Kessler. 1997, Angew. Chem. Int. Ed. Engl. ; (36) pp. 1374-1389).
The role of RGD sequence is described, for example, in Grant et al., J. Cell Physiology, 1992, Saiki et al., Jpn. J. Cancer Res. 81; 668-75. Carron et al, 1998, Cancer Res. 1; 58(9):1930-5 disclosed an RGD-containing tripeptide, named SC-68448, capable of inhibiting the binding between xcex1vxcex23 integrin with vitronectin (IC50=1 nM). Other works (Sheu et al., 1197, BBA; 1336(3):445-54xe2x80x94Buckle at al., 1999, Nature 397:534-9) showed that RGD peptides can diffuse through the cell membrane and bind to the protein caspase-3, inducing apoptosis.
Therefore, RGD sequence is the basis for developing antagonists of the different integrins. To date, the reasons for which in many cases a high selectivity for certain integrins is observed is not quite clear, although a different conformation of the RGD sequence can be taken as an explanation. Recent data demonstrated that this sequence is often inserted into a type II-xcex2-turn between two xcex2-sheets extending from the core of the protein.
Thus the problem to provide substances having high selectivity toward integrins has not been fully satisfied yet.
There is a structural constraint to this research, namely, the RGD sequence must be kept unaltered, since it is well known that any modification to this sequence implies a loss of activity.
To find the correct structure that can block the molecule in a precise reverse-turn conformation, inducing a xcex2-turn geometry, is very critical.
It is well known that the xcex1vxcex23-receptor, a member of the integrin family, is implicated in angiogenesis and in human tumor metastasis.
Metastasis of several tumor cell lines as well as tumor-induced angiogenesis can be inhibited by antibodies or small, synthetic peptides acting as ligands for these receptors (Friedlander et al.: Science 1995, 270, 1500-1502.
In order to have an inhibiting property, all the peptides must contain the Arg-Gly-Asp (RGD) sequence. Notwithstanding this RGD sequence, a high substrate specificity is present, due to different conformations of the RGD sequence in different matrix proteins (Ruoshlati et al. Science 1987, 238, 491-497). This flexibility of particular RGD portion is an obstacle to the determination of the bioactive conformation to be used in the widespread structure-activity drug design.
A solution was provided by Haubner et al. (J. Am. Chem. Soc. 1996, 118, 7881-7891) by inserting the RGD sequence in cyclic, rigid peptide structure. Spatial screening led to the highly active first-generation peptide c(RGDfV) (cyclic Arg-Gly-Asp-D-Phe-Val; WO97/06791), which shows a xcex2IIxe2x80x2/xcex3-turn arrangement. A reduction of the flexibility is a technical goal to be achieved in order to obtain antagonists of integrins. Due to the width of the integrin family and to the number of different physiological activities of said integrins, it is highly desired to obtain active agents having highly selective inhibiting action.
A solution proposed in the art was to introduce in the peptido-mimetic structure a rigid building block (turn mimetics).
Despite different tentatives and a number of structures proposed, Haubner et al. (J. Am. Chem. Soc. 1996, 118, 7881-7891), identified an RGDxe2x80x9cspiroxe2x80x9d structure capable of providing the desired xcex2IIxe2x80x2/xcex3-turn arrangement. Actually, four different structures are enabled in this work: an (S)-proline derivative, an (R)-proline derivative, a thiazabicyclo structure and a diaza-spiro-bicyclic structure. Non-homogeneous results were obtained. The spiro structure was the only one able to adopt a xcex2IIxe2x80x2/xcex3-turn conformation, but lacks of biological activity. The (R)-proline is very active, but less selective. The (S)-proline is active and selective. The thiazabicyclo-structure is active, but has the disadvantage to be less selective.
WO91/15515 discloses cyclic peptides, also containing the RGD sequence, useful for treating thrombosis, through the selective inhibition of the platelet aggregation receptor GPIIb/IIa.
WO92/17492 discloses cyclic peptides, also containing the RGD sequence, useful for treating thrombosis, through the selective inhibition of the platelet aggregation receptor GPIIb/IIa. These peptides contain also a positively charged nitrogen containing exocyclic moiety stably bonded to the cyclic peptide through a carbonyl. No beta-turns are contained in these structures.
WO94/29349 discloses a long peptide containing a -Cys-S-S-Cys-cyclic portion for the treatment of a venous or arterial thrombotic condition. This trifunctional peptide combines both catalytic and anion binding exosite inhibition of thrombin with GP IIb/IIIa receptor inhibition.
Other peptides active in treating thrombosis are disclosed in WO95/00544.
WO97/06791 discloses the use of c(RGDfV) as selective inhibitor of xcex1v/xcex35 and useful as inhibitor of angiogenesis.
WO97/08203 discloses circular RGD-containing peptides, which comprise the motif (/P)DD(G/L)(W/L)(W/L/M).
U.S. Pat. Nos. 5,767,071 and 5,780,426 disclose non-RGD amino acid cyclic peptides binding xcex1v/xcex33 integrin receptor.
U.S. Pat. No. 5,766,591 discloses RGD-peptides for inhibiting xcex1v/xcex33 receptor and useful as antiangiogenesis agents. No beta turn portions are taught.
WO98/56407 and WO98/56408 disclose fibronectin antagonists as therapeutic agents and broad-spectrum enhancers of antibiotic therapy. Said fibronectin antagonists bind to a xcex15xcex21 integrin to the purpose to prevent intracellular invasion by microbial pathogens. Some of these inhibitors are linear or cyclic peptides containing the RGD structure or antibodies. Integrin antagonists are specifically disclosed for their selectivity against xcex15xcex21 integrin. The best of them proved to be (S)-2-[2,4,6-trimethylphenyl)sulfonyl]amino-3-[[7benzyloxycarbonyl-8-(2-pyridinylaminomethyl)-1-oxa-2,7-diazaspiro-[4,4]-non-2-en-3-yl]carbonylamino]propionic acid.
U.S. Pat. No. 5,773,412 discloses a method for altering xcex1vxcex23 integrin receptor-mediated binding of a cell to a matrix, said cell being an endothelial or smooth muscle cell, by contacting said cell with a RGD-containing cyclic peptide. Also disclosed there is a method for inhibiting angiogenesis by using this cyclic peptide. The cyclic peptide disclosed in U.S. Pat. No. 5,773,412 contains at least 6 amino acids and the RGD sequence is flanked, on the D-side, by a first amino acid which can provide a hydrogen bond interaction with an integrin receptor (Asn, Ser or Thr) and a second amino acid, that has the characteristics of hydrophobicity or conformational constraint (Tic, i.e. 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, Pro, Phe or Ile). A selection of these peptides are taught as useful for altering the binding of osteoclasts to a matrix such as bone or for selectively altering integrin receptor binding. It has now been found that cyclic pseudopeptides having an RGD mimetic structure characterized by an azabicycloalkane structure are endowed with selective inhibition of xcex1vxcex23 integrin-mediated cell attachment. This activity makes them useful as therapeutical agents, in particular for treating pathologies due to an altered angiogenesis, for example tumors.
It is an object of the present invention, compounds of formula (I) 
wherein
n is the number 0, 1 or 2,
Arg is the amino acid L-Arginine, Gly is the amino acid Glycine and Asp is the amino acid L-Aspartic acid, and the pharmaceutically acceptable salts thereof, their racemates, single enantiomers and stereoisomers.
The compounds of formula (I) are selective inhibitors of xcex1vxcex23 receptor. Accordingly, they are useful for treating all those pathologies due to an altered xcex1vxcex23 integrin-mediated cell attachment; for example, retinopathies, acute renal failure, osteoporosis, tumors, also associated with metastasis. The compounds of the present invention can be considered as antiangiogenesis agents, in particular for the treatment of tumors, comprising tumors associated with metastasis.
Other objects of the present invention are processes for the preparation of the compounds of formula (I).
A further object of the present invention is a method for treating a subject, whether human or animal, suffering of a tumor, by inducing an inhibition of angiogenesis, in particular for inhibiting or reducing or blocking metastatic proliferation, with the administration of a therapeutic or preventive dose of at least a compound of formula (I). Also objects of the present invention are: a method for selectively inhibiting xcex1vxcex23 integrin-mediated cell attachment to an RGD-containing ligand, comprising contacting said ligand with an effective amount of a compound of formula (I); a method for treating a subject suffering from a pathology related to an altered xcex1vxcex23 integrin-mediated cell attachment comprising administering to said subject a compound of formula (I); said pathologies being for example retinopathy, acute renal failure, osteoporosis.
From the industrial application point of view, the present invention also comprises pharmaceutical compositions comprising an effective dose of at least a compound of formula (I) in admixture with pharmaceutically acceptable vehicles and/or excipients.